The inflammasomes are crucial innate immune signaling platforms implicated in immune defense against infections and autoimmune/autoinflammatory disorders. Despite their important physiological and pathological functions, the critical need for inflammasome-specific chemical probes has not been met and none of the reported inflammasome inhibitors directly engage the inflammasome receptor/sensor proteins. This not only impedes the progress of mechanistic studies in the inflammasome field, but also hampers the development of specific and potent therapeutics for inflammatory disorders, such as systemic lupus erythematosus (SLE), psoriasis, cryopyrin-associated periodic syndromes (CAPS), gout, type 2 diabetes, Alzheimer's disease, and atherosclerosis. My lab has made major contributions to our understanding of the structure and function of the AIM2 and IFI16 inflammasomes: we established the structural basis of sequence-independent dsDNA recognition by AIM2 and IFI16, characterized the autoinhibition mechanism that regulates the AIM2 receptor, elucidated the signaling mechanisms mediated by the pyrin, CARD and TIR domains from the inflammasome receptors and signaling adapters. We will leverage our expertise and experience and test the hypothesis that chemical compounds that bind the inflammasome receptors/sensors or adapter at critical domain interfaces will stabilize different conformational states of their structures and thus modulate ther functions. To this end, we will explore a two-pronged approach to identify chemical probes specific for the AIM2 and NLRP3 inflammasomes. In aim 1, we will perform structure-based virtual screening for chemical probes that specifically bind AIM2, NLRP3 or ASC. We anticipate that small molecules that bind and stabilize the intramolecular domain-domain interfaces and the autoinhibited structures of AIM2 and NLRP3 may serve to suppress their activation. On the other hand, disruption of the ASC PYD oligomerization interface using chemical compounds that competitively inhibit the ASC filament formation may also inhibit inflammasome activation. In aim 2, we will carry out three independent in vitro assays that allow us to screen and validate inflammasome-specific chemical compounds that bind and regulate the ASC polymerization and speck formation. These assays include the thermofluor assay and the ASC polymerization assay using purified AIM2, NLRP3 and ASC proteins, and the ASC speck formation assay using a bone marrow-derived macrophage cell line stably expressing fluorescent cerulean-tagged ASC protein. We anticipate that chemical probes that specifically modulate the activities of either the AIM2 inflammasome, the NLRP3 inflammasome, or both through ASC, will be identified and validated. Importantly, the success of this project will fulfill unmet needs for hig quality and novel chemical tools to probe the function of specific inflammasomes, as well as to stimulate the development of specific and potent therapeutics against inflammatory disorders.